5-hydroxymethylcytosine (5hmC) was first reported in cytosines of bacteriophage in 1952, and this kind of modification was also found in mammalian cells recently, for instance, neurons and embryonic stem cells of mouse. Over the last few years, a lot of researches have been done in revealing the possible role 5hmC plays in genome organization and stem cell differentiation, and TET protease family has been proved to be involved in oxidizing 5mC to 5hmC.
Though 5hmC modified base has found long before, there is no effective enzyme or chemical methods to specifically identify it and to detect its distribution in the genome. Methylation-dependent restriction endonucleases MspJI family or McrBC cannot distinguish 5mC from 5hmC, in addition, methylation-sensitive restriction endonucleases, such as MspI and HpaII, share a same behavior when reacting with both 5mC and 5hmC in most cases. Meanwhile, bisulfite sequencing analysis, which is regarded as a golden standard for DNA methylation analysis, still cannot effectively discriminate between 5mC and 5hmC. With the development of 5hmC specific antibodies, immunology-based 5hmC detecting methods, for example, dot blotting analysis, immunofluorescence and immunohistochemistry have been applied to the researches regarding hydroxymethylation modification. However, these methods, which are able to detect 5hmC's existence and expression level in a cell or a tissue, still cannot locate 5hmC's genome-wide distributions. By far, the main strategy for detecting genome-wide distribution patterns of 5hmC comprises the steps of enrichment, capturing and sequencing, for example, hMeDIP, anti-CMS and JBP-pull down, such methods comprising the enrichment-capturing step still cannot obtain the distribution of 5hmC at a single-base resolution level, meanwhile, such antibody/protein-dependent methods are also restricted because of non-specific capture or capture bias.
As discussed above, a large-scale detection technique for detecting accurate distributions of 5hmC in DNA is required, and a method for accurately detecting hydroxymethylation modification is urgently needed. This method will be an important tool in the researches about genome-wide distribution of 5hmC and relevant epigenetic regulation mechanisms, furthermore, this method will be a useful tool in studying the roles of 5hmC in disease occurrence and individual development.